Development of serological and molecular tests for herpesvirus exposure detection in tortoises.

摘要

Here is reported the development and validation of serological and molecular diagnostic tests for herpesvirus exposure detection in tortoises.; An enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to a pathogenic herpesvirus associated with an upper respiratory tract disease in Mediterranean tortoises was statistically as reliable as an established serum neutralization test (SN), universally considered the gold standard for serological diagnosis of virological infections.; An indirect immunoperoxidase assay (IIP) for the detection of antibodies (IgY) directed against a pathogenic herpesvirus in tortoise plasma and a direct immunoperoxidase assay (DIP) for the detection of herpesvirus antigen in formalin-fixed infected cell cultures and in paraffin-embedded formalin-fixed tissues were developed successively to complement the ELISA and the SN.; A polymerase chain reaction (PCR) and a reverse transcription-PCR (RT-PCR) were then developed on the basis of the sequencing information derived from the genome of an American herpesvirus isolate (HV 1976).; The serological and molecular diagnostic tests were validated with a transmission study that consisted of the experimental infection and successive challenge of a group of Greek tortoises (Testudo graeca) with pathogenic herpesviruses. Serological, histological, clinical and molecular aspects of the stomatitis-rhinitis in tortoises were investigated. An extensive molecular investigation conducted with PCR and RT-PCR tests demonstrated the systemic distribution of the herpesviral agent and its strong neurotropism. The recovery of the herpesviral isolates following the challenge was unsuccessful. However, the data collected in this study, strongly indicated tortoise herpesvirus as one of the primary causative agents of stomatitis-rhinitis in Greek tortoises. To acquire genetic information about this novel tortoise herpesvirus, three contiguous Hind III fragments of 8.667 kb in length were sequenced and analyzed. The UL 36, UL 37, UL 38, UL 39 and UL 40 tortoise herpesvirus homologues of herpes simplex virus 1 (HSV1) open reading frames were identified and fully or partially sequenced. The amino acid sequence encoded by the tortoise herpesvirus UL 39 homologue of HSV1 was used to perform a phylogenetic analysis that showed tortoise herpesvirus to belong to the sub-family of the alpha Herpesvirinae .